IL-33通过促进PPAR-β转录抑制脂蛋白脂肪酶改善心脏脂毒性机制研究

Fatty acids (FAs) are the main source of energy and the lipids origin for heart. Although they are of major importance to heart, excessive uptake of FAs causes lipid overload or cardiac lipotoxicity (CL) and may compromise cardiac function, possibly leading to cardiomyopathy. Accumulations of triglyceride (TG) and other lipids and metabolites are the pathological basis of CL. Most of the FAs taken by heart are derived from lipoprotein lipase (LPL) dependent hydrolysis of circulating triglyceride-rich lipoproteins. It has been demonstrated that excessive LPL plays a part in CL. Peroxisome Proliferator-activated receptor-β (PPAR-β) has been shown to inhibit LPL and ameliorate CL in vivo. Recently, IL-33 has been found to reduce TG accumulation in both macrophages and white adipose cultures. Besides, it is shown to up-regulate PPAR-β in liver. Our preliminary study showed a beneficial effect of IL-33 in reducing TG in cardiomyocyte cell line, accompanied with increased PPAR-β mRNA level and decreased LPL. The associations among IL-33, PPAR-β, LPL and CL are unknown. We proposed a novel mechanism that IL-33 up-regulated PPAR-β, which in turn inhibited LPL and lowered FAs uptake and finally ameliorated CL. To demonstrate these, we used our α-MHC-PPARγ transgenic mice, which occur excessive FAs uptake and severe CL in adulthood. By in vivo study, we would explore the effects of IL-33 on CL and on the expression profiles of PPAR-β and LPL. With the help of self-complementary double strain adeno-associated virus serotype 9 (dsAAV9), we are going to up-regulate or down-regulate PPAR-β and LPL, both in vivo and in vitro, demonstrating the roles of PPAR-β and LPL played in the process of IL-33-mediating anti-CL. After elucidating the roles of PPAR-β and LPL in the beneficial effects of IL-33 on CL, our next aim is to investigate the precise mechanism of IL-33 regulating PPAR-β in cardiomyocytes. Since the transcriptional factor, CCAAT/enhancer binding protein-alpha (C/EBP-α), which is down-regulated by IL-33, has been shown to inhibit transcriptional activity of PPAR-β promoter in keratinocytes, we sought to use dsAAV9 again to increase or decrease C/EBP-α expression, exploring their effects on PPAR-β expressions and demonstrating the role of C/EBP-α in the regulatory processing of IL-33 on PPAR-β. After a determination of the transcriptional mechanism of IL-33 on PPAR-β by using the actinomycin-D, we would explore the precise mechanism that C/EBP-α acts on PPAR-β promoter using dual-luciferase reporter gene analysis, EMSA and CHIP. Our works would help to elucidate the effects of IL-33 on CL and its underline mechanism, which may help to prevention and treat CL in clinical practice.

心脏脂毒性（CL）的致病基础是甘油三酯（TG）等脂毒性物质堆积，而心肌细胞过度摄取脂肪酸是其最重要原因。脂蛋白脂肪酶（LPL）是影响脂肪酸摄取的关键酶。过氧化物酶体增殖物激活受体-β（PPAR-β）已被证实可在心肌细胞内抑制LPL且对CL有益。近年针对不同细胞研究均显示白细胞介素-33（IL-33）可减少细胞内TG含量；此外又发现IL-33可上调肝脏PPAR-β。我们前期实验发现IL-33减少心肌细胞内脂质含量，同时上调PPAR-β及抑制LPL。IL-33调控PPAR-β及LPL并改善CL机制不清。本项目拟利用本单位已有自发出现过度摄取脂肪酸和CL的转基因小鼠，通过实验观察IL-33对CL的作用及其对心肌细胞表达PPAR-β和LPL的影响；通过上调或下调PPAR-β及LPL表达，探讨它们是否参与介导IL-33抗CL过程；最后通过双荧光素酶报告基因、定点突变、EMSA及CHIP等方法阐明IL-33调控PPAR-β的具体机制，为临床防治CL提供更多思路。

（一）IL-33抑制心肌脂毒性作用及机制（未发表资料）..IL-33是一种心肌保护蛋白，具有抑制心肌细胞凋亡的作用。心肌细胞过度摄取脂肪酸是心肌病变过程中重要且普遍的现象，参与了各种原因导致的心肌细胞损伤的过程。IL-33虽然被证实可减少心肌凋亡，但它对心肌细胞摄取脂肪酸的作用尚不明确。..项目实施过程中，我们首先确认了IL-33具有抑制脂肪酸在心肌细胞内堆积和减少脂毒性凋亡主要途径（内质网应激）的作用。进一步研究发现，IL-33可以显著促进被饱和脂肪酸（棕榈酸）抑制的自噬小体形成及自噬流；通过对自噬的干预，证实IL-33缓解内质网应激及内质网应激途径的凋亡是通过促进自噬实现的。因此，本项目的结果首次证实了IL-33可减少脂肪酸及其衍生物在心肌细胞内堆积并抑制脂毒性心肌细胞凋亡，并且初步揭示了其内在机制，可为心肌脂毒性这一普遍存在的现象提供更多防治思路。.（二）IL-33抑制泡沫细胞形成作用机制..已有研究证实IL-33可以减少泡沫细胞形成，但具体机制未知。在该项目经费的支持下，课题组进行了该部分机制的完整探讨，并已经完成论文撰写和发表（Zhang HF et al. Experimental and Molecular Medicine. 2017, 3;49(11):e388. doi: 10.1038/emm.2017.183；期刊影响因子：5.063）。..本课题组研究发现：IL-33促进胆固醇流出通道ABCA1表达，抑制胆固醇流入通道CD36表达；而且，促进IL-10水平可以逆转IL-33减少泡沫细胞形成的作用，同时逆转IL-33上调ABCA1的效应，但对CD36表达无影响。最后，我们证实了IL-33通过激活STATA3促进IL-10，并鉴定出在该调控过程中的核心转录元件。.（三）IL-33诱捕受体（sST2）可辅助诊断射血份数保留心衰（EFpHF）..经典心衰标志物NT-proBNP在EFpHF中价值受到一定局限。寻找可能补充EFpHF诊断价值的血清标志物有着重要的临床意义。..sST2已被证实在心力衰竭中具有一定诊断价值，但其独立诊断效能逊于NT-proBNP；然而，两者联合诊断效能未知。在该项目支持下，我们发现sST2联合NT-proBNP可进一步提高EFpHF诊断效能（罗年桑，张海峰等.中华心血管病杂志, 2017,45(3):198-203）。